Research Question:

To find out how the presence of bacteriophages in the soil around red or white oak trees has a correlation with the health condition of oak trees.

Rationale:

A Plaque Assay helps us determine if there is a presence of bacteriophages by adding Arthrobacter directly to the lysate. We can tell the existence of bacteriophages by checking the presence of plaques on the agar plate.  The new sample would provide a larger data size for the research, so besides the washing and Enrichment, the sample’s metadata is also very important for more insight into our research question.

Soil Metadata for (10.1.18):

% Water:

1. Weigh the dry soil with weigh boat.
2. Calculate the percent water.

Plaque Assay for Soil (10.1.18):

Materials:

• Micropipette
• Serological pipette
• Centrifuge tube(1.5ml)
• 50 ml conical tube
• LB Broth
• CaCl2(aq)
• Sample (10.1.18) Lysate
• 2x Top Agar
• Agar plate
• Arthrobacter

Procedure:

1. Set up an Aseptic zone.
2. Add 0.5 ml Arthrobacter and 10 ul Filtered Enriched Lysate to a Centrifuge tube 40 min for infection.
3. Add 2 ml of LB Broth, 22.5 ul Calcium Chloride (aq) to a tube
4. Add the infected lysate to the tube
5. Add 2.5 ml of 2x Top Agar, pipette up and down then decant the solution to the agar plate
6. Wait for 10 min to solidify (slightly shooked during) and place into the incubator.

Pick Plaque for Plaque Assay (10.1.18):

1. Picked two plaques and added to 100 ul phage buffer, respectively.
2. Vortex for 10 seconds.
3. Placed in fridge for storage.

Observations, Results & Data:

Soil Metadata results for Sample (10.1.18):

1. Soil Composition:  2 ml Clay,  2 ml Silt,  1.5 ml Sand
2. Soil pH = 6.0
3.  % water: 22.5 %

Plaque Assay (10.1.18) results:

Control plate showed five distinctive small colonies. The plaque plate showed traces of plaques, mostly looks round, approximate to mostly be around 0.01 cm in diameter, two of them were picked for dilution.

Interpretations & Conclusions:

The control plate showed contamination, even though the plaque plate showed plaques, due to the contaminations, another plaque assay running the same sample was necessary, to confirm the positive results.

Next Step:

If the plaque assay yields positive results it will further confirm the existence of plaques, and in the next lab dilution and plaque assays of dilution lysates would be the main objective.